CN-114907960-B - Label-free living cell screening system and method based on droplet microfluidic

CN114907960BCN 114907960 BCN114907960 BCN 114907960BCN-114907960-B

Abstract

The invention provides a label-free living cell screening system and method based on droplet microfluidics, wherein the system comprises a microfluidic chip, a fluorescence signal monitoring site, an imaging site and a sorting site, a fluorescence detection unit, an imaging unit, a sorting unit and a signal processor, wherein the microfluidic chip is provided with the fluorescence signal monitoring site, the imaging site and the sorting site, the fluorescence detection unit is configured to excite a fluorescence signal at the fluorescence signal monitoring site and collect the fluorescence signal, the imaging unit is configured to collect images of droplets wrapping target cells at the imaging site, the sorting unit is configured to sort out the droplets wrapping the target cells at the sorting site, the signal processor is used for triggering the imaging unit to collect the images of the droplets at the imaging site when the fluorescent signal identifies that the droplets pass through the fluorescence signal monitoring site, and the signal processor is used for triggering the sorting unit to sort out the droplets wrapping the target cells at the sorting site when judging that the droplets wrap the target cells based on image analysis. The invention monitors the liquid drops by using fluorescent signals, and improves the monitoring efficiency and accuracy, thereby greatly improving the precision and flux of liquid drop sorting.

Inventors

HUANG SHUQIANG
LI SIHONG
YU YUE
LIU CHENLI
FU XIONGFEI
ZHOU LEI
WANG JINJUAN
WEN HUI
SHEN YAXIN

Assignees

中国科学院深圳先进技术研究院

Dates

Publication Date: 20260505
Application Date: 20220520

Claims (10)

1. A label-free living cell screening system based on droplet microfluidic, comprising: The microfluidic chip comprises a microfluidic channel and is configured to be used for conveying liquid drops, wherein fluorescent signal monitoring sites, imaging sites and sorting sites are sequentially arranged on the microfluidic channel; A fluorescence detection unit configured to emit excitation light to the fluorescence signal monitoring site to excite a fluorescence signal carried by a droplet, and collect the fluorescence signal; an imaging unit configured to collect an image of a droplet at the imaging site with illumination light emitted therefrom, the illumination light and the excitation light having wavelengths different from each other; a sorting unit configured to sort out droplets wrapping the target cells at the sorting location; The signal processor is respectively connected with the fluorescence detection unit, the imaging unit and the sorting unit; The signal processor triggers the imaging unit to collect images of the liquid drops at the imaging sites when the signal processor recognizes that the liquid drops pass through the fluorescent signal monitoring sites based on the fluorescent signals, and triggers the sorting unit to sort out the liquid drops wrapping the target cells at the sorting sites when judging that the liquid drops wrap the target cells based on the images, wherein the target cells are label-free target cells.
2. The label-free living cell screening system according to claim 1, wherein the microfluidic channel comprises a main transport channel, a first injection channel and a second injection channel connected to a first end of the main transport channel, and a first output channel and a second output channel connected to a second end of the main transport channel, wherein the fluorescent signal monitoring site, the imaging site, and the sorting site are sequentially disposed on the main transport channel in a direction from the first end to the second end, and wherein the sorting site is disposed adjacent to an intersection of the first output channel and the second output channel.
3. The label-free living cell screening system according to claim 2, wherein a fluid driving device is connected to the microfluidic chip, the fluid driving device being configured to inject a liquid droplet into the first injection channel and a continuous liquid phase into the second injection channel, and to drive the liquid droplet to flow from the first end toward the second end of the main transport channel.
4. The label-free living cell screening system according to claim 3, wherein the fluorescence detection unit comprises an excitation light module and a detection module, the excitation light module comprises a light source and a beam shaping element, the detection module comprises a converging lens and a high-speed detector, wherein the excitation light emitted by the light source enters the fluorescence signal monitoring site through the beam shaping element to excite fluorescence, the excited fluorescence enters the high-speed detector through the converging lens in a converging way, and the high-speed detector sends the collected fluorescence signal to the signal processor.
5. The label-free living cell screening system according to claim 3, wherein the imaging unit comprises an illumination module, an objective lens, an imaging lens and a high-speed imaging camera, the illumination module and the objective lens are located on opposite upper and lower sides of the microfluidic channel, illumination light emitted by the illumination module is collected by the objective lens, focused on the high-speed imaging camera via the imaging lens, and the high-speed imaging camera sends collected image signals to the signal processor.
6. The label-free living cell screening system according to claim 3, wherein the sorting unit includes a voltage amplifier and an electrode, the electrode being disposed in the microfluidic chip, the electrode being located on a side of the main transport channel and adjacent to the sorting site, the voltage amplifier applying a voltage to the electrode under the control of the signal processor to generate a non-uniform electric field, thereby sorting out droplets wrapping target cells.
7. A method for label-free living cell screening based on droplet microfluidic, characterized in that the label-free living cell screening system according to any one of claims 1-6 is used, the method comprising the steps of: driving liquid drops along with continuous liquid phases to flow from a first end to a second end in a microfluidic channel of the microfluidic chip; Controlling the fluorescence detection unit to emit excitation light to the fluorescence signal monitoring site so as to excite a fluorescence signal of the liquid drop, collecting the fluorescence signal and feeding back to the signal processor; The signal processor judges whether the liquid drops pass through the fluorescent signal monitoring site according to the fluorescent signal, if so, the imaging unit is triggered to acquire images of the liquid drops at the imaging site and feeds the images back to the signal processor; and the signal processor judges whether the liquid drops wrap target cells or not according to the images of the liquid drops, if so, the sorting unit is triggered to sort out the liquid drops wrapping the target cells at the sorting position.
8. The method of label-free living cell screening according to claim 7, wherein said determining whether a droplet has passed the fluorescent signal monitoring site comprises: And the signal processor continuously receives the fluorescent signal sequence fed back from the fluorescent detection unit, compares the fluorescent signal sequence with a set fluorescent signal threshold value, and judges that a liquid drop passes through the fluorescent signal monitoring site when the signal value of the fluorescent signal sequence firstly jumps from being lower than the threshold value to being higher than the threshold value and generates a rising edge, and then jumps from being higher than the threshold value to being lower than the threshold value and generates a falling edge.
9. The method of claim 7, wherein determining whether the target cells are encapsulated within the droplet based on the image of the droplet comprises: performing background filtering on the collected original image of the liquid drop; Processing the filtered image by using a morphological operator, and identifying the boundary of the liquid drop and cells in the liquid drop to obtain mask morphological data of the cells; and calculating and obtaining the phenotype characteristics of the cells marked by the mask in the liquid drop, comparing the phenotype characteristics with preset conditions, and judging that the liquid drop wraps the target cells if the phenotype characteristics meet the preset conditions.
10. The method of label-free living cell screening according to claim 7, wherein triggering the sorting unit to sort out droplets of packed target cells at the sorting location comprises: The signal processor controls the sorting unit to generate a non-uniform electric field at the sorting site, so that the flow path of the liquid drops wrapping the target cells is deviated, and the liquid drops wrapping the target cells are controlled to flow from a preset output channel, so that the liquid drops wrapping the target cells are screened out.

Description

Label-free living cell screening system and method based on droplet microfluidic Technical Field The invention relates to the technical field of cell sorting, in particular to a label-free living cell screening system and method based on droplet microfluidic. Background The label-free living cell screening refers to that the target cells are separated from one population and used for subsequent culture, analysis and utilization under the premise of ensuring the activity of the cells only according to the physiological phenotype characteristics of the cells without any pretreatment of the cells. The label-free living cell screening has very wide application prospect in the field of biology, and particularly has great application potential in screening microbial cells with important industrial or medical value. Droplet microfluidics is considered to be one of the best technological approaches to achieve label-free living cell screening, mainly because a large number of microdroplets can provide independent growth spaces for each cell in a population, cells grow in the respective spaces and exhibit physiological phenotypes, large molecular proteins or small molecular compounds secreted by the cells are confined within the respective droplet spaces, and interference between cells is completely eliminated, making it easier for cells with specific phenotypic characteristics to be identified and screened. Currently, label-free living cell screening methods based on droplet microfluidic can be classified into five types according to cell phenotype detection principles, namely light absorption/scattering method, raman spectroscopy, mass spectrometry, electrochemical method and imaging method. The Raman spectroscopy, the mass spectrometry and the electrochemical method can only detect cell metabolites, so that the application range is limited, the light absorption/scattering method can only detect cell quantity changes, the application range is also very limited, and the imaging method can detect various physiological phenotype characteristics including cell morphology, quantity and metabolic activity and has wider application prospect. Therefore, the development of the imaging-based droplet microfluidic label-free living cell screening technology is significant. The imaging-based droplet microfluidic label-free living cell screening method generally comprises the following steps: (1) Generating micro-droplets with uniform size by using a microfluidic chip, and wrapping single or multiple cells into the micro-droplets while generating the micro-droplets; (2) Collecting and incubating the microdroplets to grow cells within the microdroplets and exhibit a physiological phenotype; (3) Injecting the micro-droplets into a microfluidic droplet sorting chip, collecting images of each micro-droplet, and identifying target cells by analyzing physiological phenotype characteristics of cells in the micro-droplets; (4) After the target cells are identified, the micro-droplets wrapped with the target cells are separated, and the target cells are obtained by recovering the separated micro-droplets, so that the purpose of cell screening is finally achieved. The core point of the method is the micro-droplet image acquisition and the physiological phenotype characteristic analysis of the cells in the step (3). The quality of the micro-droplet image and the performance of the physiological phenotype characteristic analysis of the cells directly influence the sensitivity and the precision of the cell screening. At present, a series of micro-droplet image acquisition and cell physiological phenotype characteristic analysis methods have been proposed. These methods can be classified into two types according to the image acquisition mode: 1. The continuous image acquisition method comprises the steps of continuously acquiring images of a microfluidic chip channel at a fixed frequency by using a high-speed camera, analyzing whether micro-droplets flowing through the images are in each acquired image in real time, further analyzing physiological phenotype characteristics of cells in the droplets when the micro-droplets are in the acquired images, judging whether the cells in the micro-droplets are target cells, and executing a droplet sorting function to recover the target cells after judging that the cells are target cells. In the continuous image acquisition method, most of the images acquired by the high-speed camera are invalid data, namely image data of liquid drops which are not shot. However, in order to find an image of a captured droplet, these invalid data still need to be analyzed one by one, and image analysis is a rate limiting step of the whole technical method, and analysis of a large amount of invalid image data seriously affects the efficiency of performing droplet sorting while limiting the screening flux of cells. In addition, analysis of invalid data wastes a lot of hardware resources, and the equipment cost is higher on